1. Field of the Invention
The present invention relates to a method of analyzing and measuring mRNA of genes which specifically express in gastric cancer cells among red blood cells, lymphoid cells, detached intraperitoneal mesothelial cells and a very small amount of free gastric cancer cells contained in a sample obtained from peritoneal wash and the like specimen from a patient before or immediately after procedures such as removal of gastric cancer. Specifically, the present invention relates to an oligonucleotide probe for detection, a probe set and a support carrying the probe. The present invention also relates to a primer and a primer set for amplifying the genes as well as a method of detecting of a gene using the same. Further, the present invention relates to a method of acquiring information to predict postoperative recurrence of gastric cancer with high sensitivity and high accuracy by measuring the gene expression level peculiar to cancer cells.
2. Related Background Art
In the therapy of cancer, recurrence after surgical or endoscopic resection of primary tumor or radiation therapy, chemotherapy and chemoradiotherapy is an important matter of life-and-death matter for a patient. The death by metastasis/recurrence is overwhelmingly more frequent than the death resulted from primary tumor in the death by carcinoma as a main cause. Accordingly, prediction of recurrence to a patient with cancer from whom the primary tumor has been removed particularly by surgical resection will be not only an important pointer in designing the future of the patient with cancer but also critically important for deciding a treatment plan such as postoperative chemotherapy.
It is usually desired in a patient after resection of gastric cancer to monitor the recurrence for a long term by periodic medical examination mainly by postoperative diagnostic imaging. However, when there is no abnormality in one year or more, the long-term monitoring is not always achieved, and there are many cases in which recurrence suddenly occurs afterwards. In the meantime, observation of cells which can be sampled in abdominal operation before resection, that is check of cancer cells by cytological diagnosis by a pathologist, is also performed. This is, for example, a diagnosis method taken at the time of an operation of gastric cancer (perioperative quick cytological diagnosis). When the existence of free cancer cell is recognized, even though in a small number, in physiologically existing intraperitoneal free cells (mainly, red blood cells, lymphoid cells, detached intraperitoneal mesothelial cells), progression of cancer generally advances, and metastasis and recurrence occur at a high probability. The cytological diagnosis is positioned as an extremely important testing method for cancer not only for inferring the prognosis of a patient but also selecting intraoperative and/or postoperative auxiliary chemotherapies (administration of an anticancer drug, hyperthermia treatment, etc.) from the results thereof.
As commonly performed cytological diagnosis methods, there is a method in which cells applied on a glass slide from peritoneal wash are subjected to Papanicolou staining and the shape thereof is observed (simple cytological diagnosis) and a method in which proteins referred to as tumor markers such as CEA (carcinoembryonic antigen) are subjected to immunostaining (immunological cytological diagnosis). The latter, immunological cytological diagnosis, is slow in spreading because it incurs labor and cost. On the other hand, the former, simple cytological diagnosis, is normally performed in Japan on the occasion of a gastric cancer operation. However, identification of an extremely small amount of cancer cells as few as around 1 to 5 per one piece of glass slide by observation with a microscope is practically almost impossible, and the diagnosis by the shape largely depends on the subjectivity of a pathologist and needs considerable skill in the diagnosis. Besides, pathologists performing the diagnosis are chronically short in number, and the specialized domain is subdivided. Therefore, it is practically difficult to allocate the pathologists who can perform the cytological diagnosis at the same level in the majority of hospitals, and a more objective evaluation method is demanded.
Techniques aiming at discerning various cancer markers at a gene expression level as diagnosis technology to meet with such needs have been suggested. This is, for example, determination by real time polymerase chain reaction of mRNA of CEA which serves as a marker in immunostaining. However, when expression of a single gene is utilized as information, there is variation in the expression level for each case, and it has been pointed out that expression may occur in the background, i.e., in cells other than cancer cells (red blood cells, lymphoid cells, detached intraperitoneal mesothelial cells), and pseudo-positive and pseudo-negative are involved (Nagao K, Hisatomi H, Hirata H, et al. Expression of molecular marker genes in various types of normal tissue: Implication for detection of micrometastases. Int J Mol Med 2, 2002, 10: 307-310). Although this method enables to obtain better results in prognostication of gastric cancer as compared with the above-mentioned cytological diagnosis, diagnosis only with CEA has not been spread.
In addition, it has been reported by K. Mori et al./Biochemical and Biophysical Research Communications 313(2004) 931-937, that a plurality of genes other than CEA are useful for the prediction of recurrence of gastric cancer.